Methods for detecting small mutations such as single base substitutions in nucleic acids provide powerful tools for a variety of purposes, including cancer diagnosis and prognosis, perinatal screening for inherited diseases, and the analysis of genetic polymorphisms, for example for genetic mapping or identification purposes. A mutant nucleic acid that includes a single nucleotide change or multiple nucleotide changes will form base pair mismatches after denaturation and subsequent annealing with the corresponding wild type and complementary nucleic acid.
Several types of methods have been used to detect such nucleic acid mismatches, but they often exhibit drawbacks. For example, methods that depend on mismatch selective DNA binding proteins lack easy mapping capabilities. Methods based on conformation-dependent DNA electrophoretic mobility difference induced by small sequence changes, such as SSCP (single-strand conformation polymorphism) and DGGE (denaturing gradient gel electrophoresis) are widely used. They, however, are unable to show the location of mutations; and DNA length limitations and the need for optimization for individual experiments make them cumbersome ( Cotton et al. (1998), Mutation detection: a practical approach (IRL Press at Oxford University Press, New York). Other methods that use chemicals or RNAses as cleavage agents at heteroduplex sites either can detect only a subset of mutations, involve hazardous materials or require multiple steps (Myers et al (1985), Science 229, 242-7; Cotton et al. (1988), Proc Natl Acad Sci USA 85, 4397-401). Another method uses T4 endonuclease VII as heteroduplex-cleaving enzyme (Youil et al. (1995), Proc Natl Acad Sci USA 92, 87-91; Mashal et al. (1995), Nat Genet 9, 177-83).
Transposons are genetic elements that move from one location in the genome to another. The transposition process involves DNA cleavage at the 3′ ends of the transposon followed by the rejoining of the 3′OH termini to a new target DNA site (Mizuuchi, K. (1992), Annu Rev Biochem 61, 1011-51). These steps are catalyzed by the element-specific transposase proteins. Phage Mu propagates by replicative transposition that is catalyzed by the MuA transposase. While this reaction is physiologically controlled by a number of regulatory cofactors, the DNA cleavage and joining reactions can be promoted in vitro, by the transposase protein and a DNA fragment with the right end sequence of Mu genome (Savilahti et al. (1995), Embo J 14, 4893-903; Mizuuchi et al. (1989), Cell 58, 399-408; Craigie et al. (1986), Cell 45, 793-800). Mu can transpose to essentially any DNA sequence.
The inventors report herein that, unexpectedly, Mu displays a dramatic preference for insertion into mismatched DNA sites. This newly identified specificity allows for methods to detect and map mismatched DNA sites, hence genetic mutations, in the presence of a large excess of nonspecific DNA.